Tissue samples were obtained from autopsy materials that were collected at the Tokyo Metropolitan Geriatric Hospital and the Institute of Gerontology [1, 6, 10]. Among them, we analyzed all cases of cerebrovascular disease (CVD) (including cerebral infarcts or hemorrhage) with a lobar lesion. For this study, we defined a lobar infarct or hemorrhage as involving more than one-third of the MCA area. We excluded cases with brain tumors or neuroinflammatory diseases.
Three neuropathologists reviewed each case separately and then conferred to reach the final diagnosis. Clinical information was obtained retrospectively from medical charts and summaries and was reviewed by two board-certified neurologists.
Sampling and routine and immunohistochemical staining
Brains and spinal cords were examined according to our BBAR protocol [1, 6, 10]. The brains and spinal cords were fixed in 20% buffered formalin (Wako, Osaka, Japan) for 7–13 days and then dehydrated in a graded alcohol series, cleared in xylene, and embedded in paraffin. Serial sections (6 μm thick) were cut and stained with hematoxylin and eosin and by the Klüver–Barrera method. They were further examined with Gallyas Braak silver staining .
For immunohistochemistry, we used the Ventana Benchmark XT autoimmunostainer (Ventana, Tucson, AZ, USA) according to the manufacture’s protocol [1, 6, 10]. The BNM sections were immunostained using the following antibodies raised against synthetic peptide corresponding to phosphorylated tau (ptau; AT-8, 1:100, monoclonal; Innogenetics, Ghent, Belgium), phosphorylated α-synuclein (pSyn#64, polyclonal, 1:20,000; Wako, Osaka, Japan), and phosphorylated TDP-43 (pTDP43; pSer409/410, monoclonal, 1:10,000; Cosmo Bio, Tokyo, Japan). The signals from monoclonal and polyclonal antibodies were detected using the automatic system on a Ventana Discovery with the I-View DAB Universal Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Sections were counterstained with hematoxylin.
Analysis of the BNM
The cerebrum was sliced in coronal sections vertical to the anterior and posterior commissure line. The BNM sections  at the level of the anterior commissure line were immunostained using the following antibodies raised against synthetic peptide corresponding to AT8: pSyn#64, pSer409/410, anti-3-repeat tau (RD3, monoclonal, 1:2000; Merck, Darmstadt, Germany), and 4-repeat tau (RD4, monoclonal, 1:50; Merck) and (anti-4R, monoclonal, 1:500; Cosmobio). They were further examined with Gallyas Braak silver staining .
Quantitative analysis of phosphorylated tau immunoreactive (ptau+) neurons in the BNM
To obtain the number of ptau+ neurons, we counted only the number of the neurons with nucleoli. The immunoreactive density of total BNM neurons was calculated. In addition, the ratio of RD3−/RD4- or anti-4R immunoreactive neurons was calculated.
Semi-quantitative analysis of pTDP43 immunoreactive (pTDP43+) neurons in the BNM
We semi-quantitatively analyzed the immunohistochemical staining with pTDP43 antibody. Our grading system was modified based on the scoring system of a previous study : Neuronal cytoplasmic inclusions (NCIs), glial cytoplasmic inclusions (GCIs), and neuronal intranuclear inclusions (NIIs) immunoreactive for pTDP43 were quantitatively analyzed and scored on a scale of 0–3, depending on the total number of pTDP-43+ NCIs, GCIs, or NIIs: 0 = none; 1 = 1–3; 2 = 4–9; 3 = ≥10. pTDP43+ dystrophic neurites (DNs) were semi-quantitatively scored as 0–3, where 0, absent; 1, sparse; 2, moderate; 3, severe.
The sarkosyl-insoluble fractions were prepared as described by Taniguchi-Watanabe et al. . Frozen BNM (0.25 g) from one MCA territory infarct case (case 1) was homogenized in 20 volumes (5 ml) of buffer A (10 mM Tris-HCl, pH 7.5, containing 1 mM EGTA, 10% sucrose, and 0.8 M NaCl). After addition of Sarkosyl (final concentration at 2%), the homogenate was incubated for 30 min at 37 °C and spun at 20,000×g for 10 min at 25 °C. The supernatant was removed, transferred to 1.5-mL tubes,+ and ultracentrifuged at 100,000×g for 20 min at 25 °C. The pellets were washed by ultracentrifugation with 0.5 mL of sterile saline, solubilized in sodium dodecyl sulfate-sample buffer, and subjected to 4–20% gradient polyacrylamide gel (Wako) for electrophoresis. Transferred proteins on PVDF membrane was probed with the antibodies to tau T46 (Thermo) at 1:1000, RD3 at 1:500, RD4 at 1:500 and anti-4R at 1:1000, biotinylated 2nd antibody, avidin–biotin complex (Vector) and developed with diaminobenzidine and nickel chloride.
The Mann-Whitney’s U test was used to analyze differences in NFTs or pTDP43+ structures between the BNM-affected and BNM-unaffected sides. The Spearman’s rank correlation coefficient was used to analyze correlation between the numbers of anti-RD3 antibody-immunoreactive (RD3+) and anti-RD4 antibody-immunoreactive (RD4+) or anti-4R immunoreactive (4R+) neurons. Statistical analysis was performed using SPSS 15.0J software (SPSS, Chicago, IL, USA). Statistical significance was set at p < 0.05.